Fluorescence analyzing method, fluorescence analyzing apparatus and image detecting method

ABSTRACT

A fluorescence analyzing method includes the steps of irradiating a board, to which oligonucleotide is fixed, with light for fluorescence measurement; focusing produced fluorescence to form an image; and detecting the fluorescence with a two-dimensional sensor. Here, the board is provided with plural regions to which the oligonucleotide is fixed, and the plural regions are spaced apart from one another on the board substantially equidistantly in the vertical and horizontal directions. A fluorescent image is detected in a condition where the following equation is satisfied: 
     
       
      
       dd=ds×M/n  
      
     
     where ds denotes the interval between the regions, M denotes the imaging magnification of an optical focusing/imaging system, dd denotes the pixel pitch of the two-dimensional sensor, and n denotes an integer (n=1, 2, 3, 4, 5).

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/806,318, filed on May 31, 2007, claiming priority of Japanese PatentApplication No. 2006-150883, filed on May 31, 2006, the disclosures ofwhich Applications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an image detecting method. For example,the present invention relates to a method and an apparatus which capturefluorescence-labeled oligonucleotide in plural positions on a plane, andwhich then detect a fluorescence intensity and a fluorescence pattern onthe basis of the captured oligonucleotide.

2. Description of the Related Art

The techniques of analyzing DNA, RNA, protein and so on are important inthe fields of medicine, biology and the like, including gene analysisand gene diagnosis. Particularly in recent years, attention has beengiven to a method and an apparatus for simultaneously examining andanalyzing various types of DNA sequence information and geneticinformation from one analyte, by using a DNA microarray (also defined asvarious names, such as an oligochip, a DNA chip and a biochip, whichwill be hereinafter collectively called a “DNA microarray”). The DNAmicroarray is formed by using a glass board or the like, which isdivided into plural regions (e.g., several hundreds of regions toseveral tens of millions of regions), each of which is provided with atarget DNA probe (generally, the DNA probes are of different types)immobilized thereon to form a minute reaction region. When the reactionregion is caused to react with an analyte, an object DNA in the analyteis hybridized with, and captured by the immobilized DNA probe. When afluorescence probe or the like is further bonded to the target DNA,fluorescence intensity or the like can be measured to determine a bondedstate (i.e., a bonded position, which means a hybridized sequence) andthe amount of bonding. The measurement results can be utilized for genediagnosis, sequencing and so on.

An apparatus similar to a microscope (e.g., a confocal fluorescencemicroscope) generally called a “scanner” is used for reading thefluorescence intensity of the DNA microarray (e.g., JP-A No. Hei9-503308 and JP-A No. 2000-69998). This apparatus irradiates the arraywith a minute spot beam of excitation light, such as laser light, thenseparates emitted fluorescence from the excitation light by using aspectroscope such as an interference filter, and then detects thefluorescence intensity by means of a photodetector such as aphotomultiplier tube. In this event, the apparatus can determine thedistribution of fluorescence intensity throughout the array, that is,the degree of bonding to each DNA probe, by two-dimensionally scanningthe relevant minute spot formed on the array by use of a galvanometermirror or the like, or by two-dimensionally scanning the array with theposition of the minute spot fixed. Methods of reading the fluorescenceintensity of the DNA microarray include, in addition to the above beamscanning, the method for irradiating the regions of the array withexcitation light over a wide range, and then detecting a emittedfluorescent image by means of a two-dimensional camera (e.g., JP-A No.2002-181708 and JP-A No. 2001-255328). The each region having the DNAprobe immobilized is divided into several tens of sub regions, and thefluorescent intensity of the sub regions are detected. And regionsexcept DNA probe immobilized regions are divided into sub regions.

In these methods, the detection of the fluorescence on the pluralregions each having the DNA probe immobilized thereon involves makingmeasurements on the array as divided into several tens of regions, andmaking measurements on each of the regions as subdivided. This makes itpossible to detect the fluorescence on the regions as isolated from oneanother, even in a case where the plural reaction regions aremisaligned. However, the number of pixels required for thetwo-dimensional sensor is several hundreds or more times the number ofregions to be measured.

Determining of DNA, RNA sequencing is also an important technique.Samples are generally prepared in advance by labeling, withfluorescence, DNA fragments or groups of DNA fragments used insequencing, and a molecular weight pattern in separation and expansionis measured and analyzed after electrophoretic migration or inelectrophoretic migration. Specifically, a well-known Sanger method isused for inducing a dideoxy reaction, prior to the electrophoresis.Oligonucleotide with about 20 bases long, complementary to a knownportion of a base sequence of sample DNA to be analyzed, is synthesizedand labeled with a fluorophore. This oligonucleotide is used as a primerto form a complementary chain bond with about 1 picomole of the sampleDNA, and thus yielding polymerase, which in turn induces a complementarychain extension reaction. Here, four types of deoxynucleotidetriphosphates, namely, dATP, dCTP, dGTP and dTTP, as well as ddATP, forexample, are added as substrates. When ddATP is captured with thecomplementary chain extension, no further extension of the complementarychain takes place. Thus, the fragments of varying lengths terminatingwith adenine (A) are prepared. For the above reaction, ddCTP, ddGTP andddTTP are added in place of ddATP to induce reactions. The primers usedin the each reactions have the same base sequence, are labeled with fourtypes of fluorophores each of which can be spectroscopically identified.When the above four types of reactants are mixed, fragments with up toseveral hundreds of bases long and of lengths varying base by base,which fragments are complementary to the sample DNA, are obtained aslabeled with the four types of fluorophores varying depending on thetype of terminal base. The fragments are separated with a resolvingpower of one base, by capillary gel electrophoresis. The obtainedsamples migrate while being separated, and are irradiated with laser inorder from the shortest sample. When fluorescence emissions arespectroscopically measured by using plural filters, the types ofterminal bases of all fragments can be determined in order from theshortest fragment, on the basis of the temporal change in thefluorescence intensities of the respective four types of fluorescentsubstances.

Recently proposed is an approach of fixing DNA or the like to a boardfor sequencing, as described in Proc. Natl. Acad. Sci. (Proceedings ofNational Academy of Sciences), USA, vol. 100 (7), pp. 3960, 2003. Withthis approach, sequencing is performed by randomly capturing, moleculeby molecule, fragments of sample DNA to be analyzed on a surface of theboard, then inducing extension for substantially every base, and thendetecting results thereof by fluorescence measurement. Specifically,sequencing of the sample DNA is performed by repeating a cycle includingthe steps of: inducing a DNA polymerase reaction, by using four types ofdNTP derivatives (MdNTP) with detectable labels, which are captured assubstrates of DNA polymerase in template DNA, and which can terminate aDNA chain extension reaction with the presence of a protecting group;then detecting the captured MdNTP on the basis of fluorescence or thelike; and then returning the MdNTP to an extension-capable state. Sincethis technique allows sequencing of DNA fragments molecule by molecule,the technique makes it possible to concurrently analyze many fragments.Hence, analysis throughput can be increased. Since this method maypossibly make it possible to perform sequencing by DNA single molecule,the method may possibly eliminate a problem inherent in the prior art,that is, the need to refine or amplify sample DNA for cloning, PCR orthe like. Thus, speedy genome analysis and gene diagnosis can beexpected. In the method, the molecules of the fragments of the sampleDNA to be analyzed are randomly fixed on the surface of the board. Forthis reason, the method requires an expensive camera having the numberof pixels that is several hundreds of times the number of the trappedmolecules of the DNA fragments. Specifically, when the molecules of theDNA fragments are adjusted as spaced apart from one another at averageintervals of 1 micron, some molecules are spaced apart from one anotherat greater intervals, and others are spaced apart at smaller intervals.Consequently, in order to detect the molecules as isolated from oneanother, fluorescent intensities need to be detected at a minuterinterval (example, <0.1 μm). Generally, resolution of measuringfluorescent image must be several tenth of the interval between the DNAmolecules.

SUMMARY OF THE INVENTION

The aforementioned optical system requires the number of pixels, whichis several hundreds or more times the number of regions, that is, thenumber of molecules of DNA fragments where the molecules are trapped onthe board. For this reason, the optical system has a problem thatdetection speed slows down, and that an expensive two-dimensional sensoris required. Moreover, since fluorescent images need to be detected witha higher resolving power, there is another problem with increased costsfor the system in which a condenser lens with a large numerical aperture(NA) needs to be used.

An object of the present invention is to provide a method forefficiently detecting an image with a small number of pixels, such as amethod for efficiently detecting an image with a small number of pixels,when a two-dimensional sensor is used to detect a fluorescent image frommolecules of DNA fragments trapped on a board. Another object of thepresent invention is to provide a method for detecting an image at lowcosts or with easy operation, for example, when a two-dimensional sensoris used to detect a fluorescent image from molecules of DNA fragmentstrapped on a board.

The present invention relates to, for example, an image detectingmethod, in which plural targets to be measured are precisely located,and then an image of each of the plural targets is formed on a specifiedpixel of a detector including plural detecting pixels. The presentinvention provides a fluorescence analyzing method including the stepsof irradiating a board, to which oligonucleotide is fixed, with lightfor fluorescence measurement; focusing produced fluorescence to form animage; and detecting the fluorescence with a two-dimensional sensor.Here, the board is provided with plural regions to which theoligonucleotide is fixed, and the plural regions are spaced apart fromone another on the board substantially equidistantly in the vertical andhorizontal directions. Moreover, the fluorescent image is detected witha condition where the following equation is satisfied:

dd=ds×M/n

where ds denotes the interval between the regions, M denotes the imagingmagnification of an optical focusing/imaging system, dd denotes thepixel pitch of the two-dimensional sensor, and n denotes an integer(n=1, 2, 3, 4, 5).

The present invention provides another fluorescence analyzing methodincluding the steps of irradiating a board, to which oligonucleotide isfixed, with light for fluorescence measurement; focusing producedfluorescence to form an image; and detecting the fluorescence with atwo-dimensional sensor. Here, the board is provided with plural regionsto at least a part of which the oligonucleotide is fixed, and which arespaced apart from one another on the board substantially equidistantlyin the vertical and horizontal directions, and each singleoligonucleotide molecule is fixed to at least a part of the pluralregions. Moreover, the fluorescent image is detected with a conditionwhere the following equation is satisfied:

dd=ds×M/n

where ds denotes the interval between the regions, M denotes the imagingmagnification of an optical focusing/imaging system, dd denotes thepixel pitch of the two-dimensional sensor, and n denotes an integer(n=1, 2, 3, 4, 5).

More preferably, the fluorescent image is detected with the aboveequation adjusted to satisfy the following equation: dd=ds×M/n, wheren=2, 3.

The interval ds between the regions for fixing the oligonucleotidepreferably ranges between 100 nm and 10000 nm inclusive, and morepreferably, between 500 nm and 1500 nm inclusive.

Preferably, the region, to which the oligonucleotide is fixed, has adiameter of 100 nm or less.

Preferably, it is effective to form a film substance having an opticalshield function on a reaction region of a surface of the board, exceptfor the plural regions to which the oligonucleotide is fixed. A metalfilm or the like is formed by vapor deposition or the like.

Preferably, the method includes a mechanism for adjusting the imagingmagnification M to satisfy the equation, dd=ds×M/n. Moreover, registermarkers are provided respectively to at least two positions on theboard. The mechanism has a function unit for detecting the registermarkers and automatically adjusting the imaging magnification M.

The present invention makes it possible to reduce the number of pixelsrequired for the two-dimensional sensor without impairing the accuracyin measurement. For example, the number of pixels can be reduced fromseveral hundred times the number of regions to be measured to whicholigonucleotide is fixed, to thirty or less times the number of regions,or further to ten or less times the number of regions. Thus, measurementcan be performed efficiently. Accordingly, in a case where the sametwo-dimensional sensor is used, fluorescent images can be concurrentlyobtained from a larger number of regions, and thereby high throughputcan be achieved. In a case where a camera having a small number ofpixels is used, measurement can be performed more inexpensively.

For example, for the same number of molecules to be measured, thepresent invention allows efficient detection with a small number ofpixels, and hence makes it possible to reduce the price of thetwo-dimensional sensor. Moreover, optical resolution can be madesubstantially equivalent to each interval between the regions to whicholigonucleotide is fixed. Thus, a condenser lens with a large numericalaperture does not need to be used, and a low-priced lens can be used. Inaddition, it is not necessary to use an immersion lens, and thus theoperation can be made easier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a configuration diagram of a DNA examination apparatus using afluorescence analyzing method according to a first embodiment of thepresent invention.

FIG. 2 is a view for explaining the structure of a board according tothe first embodiment of the present invention.

FIG. 3 is a view for explaining correspondences in image formationbetween the board and a two-dimensional sensor according to the firstembodiment of the present invention.

FIG. 4 is a table for explaining the effects of the first embodiment ofthe present invention.

FIG. 5 is a view for explaining the structure of a board according to asecond embodiment of the present invention.

FIG. 6 is a view for explaining the structure of a board according to athird embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described below with reference toembodiments. However, it is to be understood that the present inventionis not limited to these embodiments.

First Embodiment

A description will be given of an apparatus and a method for sequencingfragments of sample DNA by equidistantly trapping, molecule by molecule,fragments of the sample DNA to be analyzed on a surface of a board;inducing extension for substantially every base; and then detecting, forone molecule at a time, captured fluorescence labels. Specifically, theapparatus and the method are used to perform sequencing of the sampleDNA by repeating a cycle including the steps of: inducing a DNApolymerase reaction by using four types of dNTP derivatives withdetectable labels, which are captured as substrates of DNA polymerase intemplate DNA, and which can terminate a DNA chain extension reaction,with the presence of a protecting group; then detecting the captureddNTP derivatives on the basis of fluorescence or the like; and thenreturning the dNTP derivatives to an extension-capable state.Incidentally, since the above operation is based on a method of singlemolecule fluorescence detection, measurement is carried out in anenvironment such as a HEPA-filtered clean room.

FIG. 1 is a configuration diagram of a DNA examination apparatus using afluorescence analyzing method according to the present invention. Theapparatus has a configuration similar to that of a normal type(epifluorescence microscope) microscope, and performs fluorescencedetection to measure an elongated state of DNA molecules trapped on aboard 8. Incidentally, the apparatus may have a configuration similar tothat of an inverted microscope.

The board 8 has a structure as shown in FIG. 2. The board 8 is made of atransparent material, for which synthetic quartz or the like can beused. The board 8 is provided with a reaction region 8 a, with which areagent or the like is brought into contact. In the reaction region 8 a,plural regions 8 ij to which DNA (oligonucleotide) is fixed. Each region8 ij has a diameter of 100 nm or less. Surfaces of the respectiveregions 8 ij are treated for trapping DNA. For example, the surfaces aretreated by bonding streptavidin to the regions 8 ij, and then causingthe regions 8 ij to react with biotinylated DNA fragments. Thereby, DNAis trapped. DNA may also be trapped by treating the surface byimmobilizing oligonucleotide of polythymidylic acid on the regions 8 ij,then treating one end of DNA fragments to yield polyadenylic acid, andthen producing a hybridization reaction. At this time, monomolecular DNAalone is allowed to enter each of the regions 8 ij by appropriatelypreparing the concentration of the DNA fragments. One molecule can betrapped in each of the regions 8 ij by making each region 8 ij smaller.Hereinafter, description will be given of measurements made on the boardunder this condition. In the board formed in such a way, there might bemonomolecular DNA in all the regions. Moreover, there might bemonomolecular DNA only on a part of the regions 8 ij. In this case, theregion 8 ij without DNA is empty. Note that an interval ds between eachregions 8 ij is set at 1 micron. A method for making the board havingthe regions spaced apart at equal intervals as mentioned above iscarried out, for example, with an approach disclosed in Japanese PatentApplication Laid-open Publication No. 2002-214142, or the like.Incidentally, the interval ds is greater than the size of each region 8ij, and is preferably about 1500 nm or less. The reaction region 8 a ofthe board has the dimensions of 1 mm×1 mm, and the number of the regions8 ij is 1000000 (=1000×1000).

“Cy3” (Trademark, Amersham) is used as the fluorescence label for dNTP.A fluorescent substance is not limited to Cy3, and various types ofother fluorescent substances may be used. In the first embodiment,measurement is carried out by using one fluorescent substance, althoughfour types of dNTP labeled respectively with different fluorescentsubstances may be used. Laser beam 1 a emitted from a apparatus 1 forexcitation of fluorescence (a YAG laser with a wavelength of 532 nm) isallowed to pass through a quarter-wave (λ/4) plate 3 to form circularlypolarized light, which then enters a quartz prism 7 for illuminationwith total reflection, through a mirror 5, a dichroic mirror 6 andanother mirror 5. Thereby, the substrate 8, on which DNA molecules areequidistantly trapped, is irradiated with the circularly polarized lightfrom the back thereof. The quartz prism 7 is in contact with the board 8with nonfluorescent glycerin interposed in between, and the laser beamis introduced to the board 8 without being reflected off the interfacebetween the quartz prism 7 and the board 8. The laser beam is totallyreflected by the surface of the board 8 at the incident angle on theboard, ranging from about 66 to 68 degrees. Thereby, evanescentillumination is formed. This enables fluorescence measurement at a highS/N ratio. A region for this laser irradiation has a diameter of about 2mm.

A temperature controller, although omitted from FIG. 1, is disposed inthe vicinity of the board. The apparatus usually has a structure whichallows illumination with halogen lamp from beneath the prism for thepurpose of observation which structure is omitted in FIG. 1.

In addition to the laser apparatus 1, a laser apparatus 2 (a YAG laserwith a wavelength of 355 nm) is disposed to allow laser to be irradiatedcoaxially with the laser beam 1 a. The laser emitted from the laserapparatus 2 is used for the step of returning the dNTP derivatives tothe extension-capable state, after performing the step of detecting thecaptured dNTP derivatives on the basis of the fluorescence.

A flow chamber 9 is disposed on top of the board 8, and is used forflowing the reagent or the like therethrough, and thereby for causingthe reagent to undergo a reaction. The chamber has a reagent inlet 12. Adispensing unit 25 having a dispensing nozzle 26, a reagent storage unit27 and a pipette tip box 28 for dispensing are used for processes suchas pouring a target reagent solution. The reagent storage unit 27 isprovided with a reagent solution container 27 a, four types ofdNTP-derivative-solution containers 27 b, 27 c, 27 d and 27 e, acleaning fluid container 27 f and so on. A dispensing tip in the tip box28 is mounted to the dispensing nozzle 26, and is then used to suck anappropriate reagent solution. The sucked solution is then introduced toa reaction region of the board through the inlet of the chamber tothereby induce a reaction. Liquid waste is discharged into a liquidwaste container 11 via a liquid drain tube 10. These operations areautomatically performed by a control PC 21.

The flow chamber is made of a transparent material along the opticalaxis, and a fluorescence image is detected, as follows. Fluorescence 13is focused by a condenser lens (an objective lens) 14 controlled with anautofocus apparatus 29. Then, fluorescence with required wavelengths isextracted with a filter unit 15. Thereby, a fluorescent image isdetected with an auto-zoom mechanism 17, an imaging lens 18 and atwo-dimensional sensor camera 19 (a high-sensitivity cooled CCD camera).The control PC 21 controls the settings of exposure time of the camera,the timing of capturing the fluorescent image, and the like, by use of atwo-dimensional sensor camera controller 20. For using four types ofdNTP, the apparatus may have such a configuration that the filter unit15 switches the relevant filters for four types of fluorescentsubstances in the time-sharing manner, and thereby detects a fluorescentimage.

For the purpose of adjustment or the like, the apparatus includes alens-barrel 16 for transmitted light observation, a television camera 23and a monitor 24, and thereby the apparatus allows observation of astate of the board 8 in real time, with halogen illumination or thelike.

As shown in FIG. 2, register markers 30 and 31 are engraved on the board8. The markers 30 and 31 are arranged parallel to the alignment of theregions 8 ij, and a distance between the markers 30 and 31 is defined.Thus, the positions of the regions 8 ij can be calculated by detectingthe markers by observation using transmitted illumination, and therebythe auto-zoom mechanism 17 can be controlled to specify a magnification.In the first embodiment, the two-dimensional sensor camera 19 (thehigh-sensitivity cooled CCD camera) having a pixel pitch (a pixel size)dd of 7.4 microns is used. When a magnification M of an imaging systemis set at ×14.8, each interval between the regions 8 ij on the board 8is divided in half for measurement.

A CCD area sensor is employed as the two-dimensional sensor camera usedin the first embodiment. Specifically, a cooled CCD camera having apixel size of 7.4×7.4 (microns) and 2048×2048 pixels is used. Thereaction region 8 a of the board has the dimensions of 1 mm×1 mm, andthe (1000×1000) regions 8 ij are spaced apart from one another atintervals of 1 micron in the reaction region 8 a. For performingmeasurement by using the cooled CCD camera with 2048×2048 pixels, themagnification M of the imaging system is set at ×14.8, and thereby eachpixel of the camera corresponds to (0.5×0.5) micron in terms of thereaction region. Besides the CCD area sensor, an image-pickup camera,such as a CMOS area sensor or the like, may be generally used as thetwo-dimensional sensor camera. Any one of two types of structures, whichare a back illuminate type and a front illuminate type, can be used forthe CCD area sensor. A CCD camera of an electron multiplication typehaving a function of signal multiplication inside an element, or thelike, is effective in improving sensitivity. It is desirable that thesensor be of the cooled type. By setting the sensor at or below about−20 degrees, dark noise of the sensor can be reduced, and thus theaccuracy in measurement can be improved.

In the first embodiment, a fluorescent image obtained from the reactionregion 8 a is detected at once. However, the image may be detected asdivided parts. When the size of the reaction region 8 a is increased tohave dimensions of, for example, 5 mm×5 mm, a single image measurementcannot cover the entire region. Thus, the image is measured as dividedinto units of 1 mm×1 mm, and then the obtained plural images arerecomposed to form the entire fluorescent image which is to be measured.In this case, an X-Y positioning mechanism for positioning the board isdisposed under a stage, and then the control PC controls movements ofthe board to the position for irradiation, light irradiation, andfluorescent image detection. In the first embodiment, the X-Ypositioning mechanism is not shown.

Various cameras can be used for measurement. In a case where a cooledCCD camera having a pixel size of 6.45×6.45 (μm) and 1392×1040 pixels isused, the magnification of the imaging system is adjusted to be 12.9. Ina case where a camera having a pixel size of 9×9 (μm) and 4008×2672pixels, the magnification of the imaging system is adjusted to be 18. Ina case where a camera having a pixel size of 16×16 (μm) and 512×512pixels, the magnification of the imaging system is adjusted to be 32.

FIG. 3 is a view for explaining correspondences in image formationbetween the board and the two-dimensional sensor according to the firstembodiment. Description will be given with a supposition that single DNAmolecules 40 (40 a, 40 b and 40 c) are trapped as spaced apart atintervals of 1 μm in a grid pattern on the board 8. Although the DNAmolecules are in fact not necessarily trapped in all positions, the sameeffect is also achieved in such a case. Thus, the description will begiven on the above supposition. An image is formed on pixels 42 of thetwo-dimensional sensor camera (the high-sensitivity cooled CCD camera)via a lens 41. For simplicity, FIG. 3 shows a situation where themagnification of the imaging system is set at 1 and the pixel pitch ofthe two-dimensional sensor camera is set at 1/14.8 (that is, thespecification is standardized with the dimensions of the board.) Thesingle DNA molecules 40 a, 40 b and 40 c correspond to pixels 42 f, 42 dand 42 b, respectively, and thereby fluorescence can be detected fromevery other pixel. As a result, the fluorescence measurement is lessaffected by overlaps of fluorescence from the respective single DNAmolecules 40 a, 40 b and 40 c, which overlaps are due to lensaberration, blurring of the image or the like, and is also less affectedby +/−10% deviation in positions where the single DNA molecules aretrapped. Thus, the measurement can be performed stably.

FIG. 4 is a table for explaining the effects. A problem exists when DNAmolecules are randomly trapped as has been conventional. Specifically,In a case of randomly trapping DNA molecules, as being performed withconventional techniques, even with the average intervals between thetrapped molecules of 1 μm, some molecules are trapped as beingstochastically close to one another while others are trapped as beingstochastically away from one another. A fluorescent image needs to bedetected at higher resolution in order to detect all these molecules asisolated from one another. However, the resolution is generallyequivalent to 0.1 μm because of constraints due to the limitation onlight diffraction, a limited number of pixels and the like. In this caseas well, it is not possible to detect all of the trapped DNA molecules,or to detect the molecules individually, and about 10% of the DNAmolecules may possibly overlap one another. Moreover, the average numberof pixels required for the camera to detect each DNA molecule is 100,which is very inefficient. In contrast, advantageous effects areachieved when DNA molecules are equidistantly spaced. Specifically, asin the case of the first embodiment, when each interval between themolecules is divided in half, that is, measurement is carried out at aresolution of 0.5 μm, it suffices that the average number of four pixelsbe used for the camera to detect each DNA molecule, and thereby themeasurement can be performed efficiently. This means that themeasurement can be performed on 25 times as many molecules as those of aconventional case, and thus high throughput can be achieved with themeasurement. In a case where measurement is performed on the samereaction region, the number of pixels to be used can be reduced, andthereby it is possible to reduce costs for the CCD. This also makes itpossible to reduce the number of pixels, and hence the cost of the CCD,when measurements are made on the same reaction region.

In the case of the first embodiment, since a numerical aperture (NA)required for the condenser lens is about 0.67 and above, a dry lens canbe used. Thus, the operation can be made easy.

Although the description has been given above of a case where eachinterval between the DNA molecules is divided in half for measurement,the same effect can be also achieved with each interval divided by 3.Furthermore, substantially the same effects can be achieved with eachinterval divided approximately by any integer between 1 and 5 inclusive.

Description will be given below of sequencing in accordance with anactual procedure for measurement. Fragments of M13-DNA are used as modelsamples. In accordance with normal practice, the end of the M13-DNAfragments is treated to form biotin. A biotinylated-DNA solution is heldin the sample solution container 27 a shown in FIG. 1, and Cy3-labeledcaged dATP, caged dCTP, caged dGTP and caged dTTP solutions (includingpolymerase) are held in the containers 27 b, 27 c, 27 d and 27 e,respectively, shown in FIG. 1. Here, the Cy3-labeled caged dNTP is acaged compound of nucleotide bonded to a 2-nitrobenzyl group. The cageddNTP is captured as a complementary chain by polymerase. Meanwhile, thecaged dNTP is inhibited from exhibiting the activity of its beingcontinuously captured by a complementary chain synthesis reaction. Forthis reason, the caged dNTP elongates by 1 base, and then stopsreacting. However, when the caged dNTP is then irradiated withultraviolet with a wavelength of 360 nm or less, a caged substance (the2-nitrobenzyl group) is liberated, and thereby the activity inherent inthe nucleotide develops to induce the next synthesis of dNTP.

With the dispensing unit 25, the biotinylated-DNA to be a template isintroduced into the flow chamber, and then is caused to react with theboard. After cleaning the chamber, an oligoprimer is introduced so thatthe primer is hybridized with the biotinylated-DNA. Thereby, acomplementary chain extension reaction occurs. After cleaning thechamber, the Cy3-labeled caged dATP solution is first introduced. When abase of the template next to the primer-bonded position is T, theCy3-labeled caged dATP is captured. After cleaning the chamber, thecaged dATP is irradiated with the laser beam 1 a (the YAG laser with awavelength of 532 nm), and then fluorescence is measured by thetwo-dimensional sensor camera. A determination can be made as to whetherthe Cy3-labeled caged dATP has been captured according to the presenceor absence of the fluorescence. Subsequently, the chamber is cleaned,and then returned to an active state by irradiating the dATP with laserbeam 2 a (the YAG laser with a wavelength of 355 nm). The aboveprocedure is executed for a cycle of the Cy3-labeled caged dCTP, cageddGTP and caged dTTP solutions. Sequencing can be accomplished byrepeating the cycle plural times.

In the first embodiment, the fluorescence-labeled caged dNTP is used asthe reagents which are the four types of dNTP derivatives with thedetectable labels captured, as the substrates of the DNA polymerase, inthe template DNA and capable of stopping the DNA chain extensionreaction, with the presence of the protecting group. Here, the reagentsmake it possible to return the dNTP derivatives to the extension-capablestate by use of certain means. However, a dNTP derivative formed ofdisulfide bonding of a fluorescent substance to nucleotide, or the like,may be used similarly perform the above sequencing. In this case, theextension stops with the presence of the fluorescent substance, while atris(2-carboxyethyl)phosphine reagent or the like can be used tochemically dissociate the disulfide bond, and thereby to return the dNTPderivative to the extension-capable state.

Second Embodiment

The apparatus according to the first embodiment is configured so thateach single DNA molecule individually enters the region 8 ij. However,the apparatus may be configured so that groups 51 of DNA molecules arespaced apart at regular intervals on a board 50, as shown in FIG. 5. Inthis case, plural molecules of the same DNA fragments are captured ineach of the region 8 ij. The base extension reaction needs almost allmolecules to be extended similarly. Therefore, the measurement needsenough reactive time. In the second embodiment, the plural moleculesexist in each of the region 8 ij. Therefore, because fluorescentstrength grows, it becomes easy to detect. Moreover, even if thereaction of some molecules doesn't proceed when tens of thousands ofmolecules are fixed to each of the region 8 ij, the influence of thesequencing on accuracy is a little. Therefore, the system can becomposed at a low cost.

Third Embodiment

Description will be given of an embodiment using another form ofreaction region. FIG. 6 shows the structure of a board 60 according tothe third embodiment. The board 60 has a structure including a reactionregion 60 a, plural regions 60 ij which are formed in the reactionregion 60 a, and in which DNA is fixed, and an optically opaque mask 60b formed around the plural regions 60 ij. Metal, such as aluminum orchromium, silicon carbide or the like, can be employed for a material ofthe mask. The material is formed into a thin film by vapor deposition orthe like. Each region 60 ij has a diameter of 100 nm or less. Methodsfor forming this opening in the mask 60 b include vapor deposition usingprojection method (with which an appropriate mask is disposed between adeposition source and the board, to be subjected to vapor deposition),electron beam lithography and direct writing using photolithography.

The third embodiment makes it possible to achieve the same effects asthose of the first embodiment. Moreover, according to the thirdembodiment, undesired stray light and fluorescence can be reduced sincethe region other than the regions 60 ij is covered with the mask. Thus,it is made possible to perform measurement at higher sensitivity.

1-10. (canceled)
 11. A fluorescence analysis apparatus for detectingfluorescence with a two-dimensional sensor by irradiating a board, towhich oligonucleotide is fixed, with light for fluorescence measurement,and then focusing the produced fluorescence to form an image,comprising: a board provided with a plurality of regions to which theoligonucleotide is fixed, and which are spaced apart from one anothersubstantially equidistantly in the vertical and horizontal directions; alight source of excitation light for fluorescence; an opticalirradiating system of excitation light for fluorescence; an opticalfocusing/imaging system for fluorescence; and a two-dimensional sensor,wherein the fluorescence analyzing apparatus is configured in a way thatthe following equation is satisfied:dd=ds×M/n where ds denotes the interval between the regions, M denotesthe imaging magnification of the optical focusing/imaging system, dddenotes the pixel pitch of the two-dimensional sensor, and n denotes aninteger (n=1, 2, 3, 4, 5).
 12. A fluorescence analysis apparatus fordetecting fluorescence with a two-dimensional sensor by irradiating aboard, to which oligonucleotide is fixed, with light for fluorescencemeasurement, and then focusing the produced fluorescence to form animage, comprising: a board provided with a plurality of regions to whichthe oligonucleotide is fixed, and which are spaced apart from oneanother substantially equidistantly in the vertical and horizontaldirections; a light source of excitation light for fluorescence; anoptical irradiating system of excitation light for fluorescence; anoptical focusing/imaging system for fluorescence; and a two-dimensionalsensor, wherein the fluorescence analyzing apparatus is configured in away that the following equation is satisfied:dd=ds×M/n where ds denotes the interval between the regions, M denotesthe imaging magnification of the optical focusing/imaging system, dddenotes the pixel pitch of the two-dimensional sensor, and n denotes aninteger (n=2, 3).
 13. A fluorescence analysis apparatus for detectingfluorescence with a two-dimensional sensor by irradiating a board, towhich oligonucleotide is fixed, with light for fluorescence measurement,and then focusing the produced fluorescence to form an image,comprising: a board provided with a plurality of regions to which theoligonucleotide is fixed, and which are spaced apart from one anothersubstantially equidistantly in the vertical and horizontal directions; alight source of excitation light for fluorescence; an opticalirradiating system of excitation light for fluorescence; an opticalfocusing/imaging system for fluorescence; a two-dimensional sensor; anda mechanism for automatically adjusting an imaging magnification tosatisfy the following equation:dd=ds×M/n where ds denotes the interval between the regions, M denotesthe imaging magnification of the optical focusing/imaging system, dddenotes a pixel pitch of the two-dimensional sensor, and n denotes aninteger (n=1, 2, 3, 4, 5).
 14. (canceled)